1. Field
Aspects of the present invention generally relate to nucleic acid analysis, and more particularly, to a system and method for determining the quantity of a particular target within a PCR reaction.
2. Description of Related Art
Polymerase Chain Reaction (PCR) is a methodology routinely used in the amplification of genetic material from template nucleotide strands or fragments. Recently, a PCR-based method known as quantitative PCR has been developed to identify target nucleotide strands or fragments in a sample population. To determine the concentration of a specific target initially present, this method utilizes a labeling dye which fluoresces in proportion to the amount of target DNA species that is produced by the PCR reaction. Two principal determinants in the accurate and reproducible quantitation of the initial target concentration are a noise assessment and a threshold value determination. The noise assessment reflects the changing reaction conditions and environment during the PCR reaction and is used to determine when target amplification is sufficiently above a background signal to enable accurate measurement of the fluorescence of the amplified target. The threshold value reflects the change in fluorescence during the PCR reaction and is typically identified as a statistically significant region above a noise baseline. Identification of the threshold value is important as it reflects the portion of the PCR reaction where a sufficient level of amplification has been achieved to allow for calculation of the initial target concentration. Conventional methods used to identify the threshold value may be subject to undesirable variability which in turn affects the accuracy of the quantitation results. Furthermore, a number of conventional threshold selection techniques rely on manual interpretation or user-based identification techniques. As a result, these methods may suffer from diminished accuracy and reproducibility. Currently, there is a need for an automated threshold selection function which overcomes the limitations associated with conventional threshold value selection techniques. Improvement in the method by which this value is identified may reduce the degree of uncertainty or variability in determining the concentration of target in the sample and improve the analytical performance of quantitative PCR.